黄芩苷通过FOXO3/CCL22信号通路调控结外NK/T细胞淋巴瘤细胞生长的机制研究

Regulation of Baicalin on Growth of Extranodal NK/T Cell Lymphoma Cells through FOXO3/CCL22 Signaling Pathway

目的:探讨黄芩苷对结外NK/T细胞淋巴瘤(ENKTCL)细胞生长的影响及相关机制。方法:培养正常NK细胞及人ENKTCL细胞株SNK-6和YTS,分别采用5、10、20μmol/L的黄芩苷处理SNK-6和YTS细胞并设置对照,EdU法检测细胞增殖,FCM法检测细胞凋亡,Western blot检测BCL-2、Bax、FOXO3和CCL22蛋白的表达;设计并合成干扰质粒,采用PEI转染试剂对FOXO3 siRNA干扰质粒和CCL22 pcDNA过表达质粒进行转染,建立动物模型并进行验证。结果:对照组和5、10、20μmol/L黄芩苷处理组SNK-6细胞的增殖率分别为(56.17±2.96)%、(51.92±4.63)%、(36.42±1.58)%、(14.60±2.81)%,各组YTS细胞的增殖率分别为(58.85±2.98)%、(51.38±1.32)%、(34.75±1.09)%、(15.45±1.10)%,各组SNK-6细胞的凋亡率分别为(5.93±0.74)%、(11.78±0.34)%、(28.46±0.44)%、(32.40±0.37)%,各组YTS细胞的凋亡率分别为(7.93±0.69)%、(16.29±1.35)%、(33.91±1.56)%、(36.27±1.06)%。与对照组相比,SNK-6和YTS细胞中BCL-2蛋白表达水平均明显降低(P<0.001),仅在黄芩苷浓度为20μmol/L时SNK-6细胞中Bax蛋白表达水平显著升高(P<0.001),而YTS细胞经不同浓度(5,10,20μmol/L)黄芩苷处理后Bax蛋白表达水平均明显升高(P<0.001)。ENKTCL细胞系SNK-6、YTS细胞较人NK细胞中FOXO3蛋白表达降低,CCL22蛋白表达升高(P<0.001),经黄芩苷处理后细胞中FOXO3蛋白表达增高(P<0.01),CCL22蛋白表达下降(P<0.001)。动物实验结果显示,黄芩苷处理可抑制肿瘤的生长,经黄芩苷处理的裸鼠ENKTCL组织中CCL22蛋白的表达水平较对照组下降(P<0.01),而FOXO3蛋白的表达水平较对照组上升(P<0.05)。此外,FOXO3沉默会导致FOXO3蛋白表达下降和CCL22蛋白表达增高(P<0.01,P<0.001)。结论:黄芩苷对ENKTCL细胞株SNK-6和YTS有抑制增殖、促进凋亡的作用,可上调Bax蛋白表达,下调BCL-2蛋白表达,且可通过FOXO3介导下调CCL22表达;动物实验发现黄芩苷可抑制肿瘤生长;黄芩苷可通过FOXO3/CCL22信号通路调控抑制ENKTCL细胞的生长并诱导其凋亡。

Objective:To investigate the effect of baicalin on the growth of extranodal NK/T cell lymphoma(ENKTCL) cells and its related mechanism.Methods:Normal NK cells and human ENKTCL cells lines SNK-6 and YTS were cultured,then SNK-6 and YTS cells were treated with 5,10,20 μmol/L baicalin and set control.Cell proliferation and apoptosis was detected by Edu method and FCM method,respectively,and expressions of BCL-2,Bax,FOXO3 and CCL22 proteins were detected by Western blot.Interference plasmids were designed and synthesized.FOXO3 siRNA interference plasmids and CCL22 pcDNA overexpression plasmids were transfected with PEI transfection reagent.Furthermore,animal models were established for validation.Results:In control group and 5,10,20 μmol/L baicalin group,the proliferation rate of SNK-6 cells was(56.17±2.96) %,(51.92±4.63) %,(36.42±1.58) %,and(14.60±2.81) %,respectively,while that of YTS cells was(58.85±2.98) %,(51.38±1.32) %,(34.75±1.09) %,and(15.45±1.10) %,respectively.In control group and 5,10,20 μmol/L baicalin group,the apoptosis rate of SNK-6 cells was(5.93±0.74) %,(11.78±0.34) %,(28.46±0.44) %,and(32.40±0.37) %,respectively,while that of YTS cells was(7.93±0.69) %,(16.29±1.35) %,(33.91±1.56) %,and(36.27±1.06) %,respectively.Compared with control group,the expression of BCL-2 protein both in SNK-6 and YTS cells decreased significantly(P<0.001),and the expression of Bax protein increased in SNK-6 cells only when the concentration of baicalin was 20 μmol/L(P<0.001),while that in YTS cells increased in all three concentrations(5,10,20 μmol/L) of baicalin(P<0.001).The expression of FOXO3 protein decreased while CCL22 protein increased in ENKTCL cell lines compared with human NK cells(P<0.001),but the expression of FOXO3 protein increased(P0.01) and CCL22 protein decreased after baicalin treatment(P<0.001).Animal experiments showed that baicalin treatment could inhibit tumor growth.The expression of CCL22 protein in ENKTCL tissue of nude mice treated with baicalin decreased compared with control group(P<0.01),while the FOXO3 protein increased(P<0.05).In addition,FOXO3 silencing resulted in the decrease of FOXO3 protein expression and increase of CCL22 protein expression(P<0.01,P<0.001).Conclusion:Baicalin can inhibit proliferation and promote apoptosis of ENKTCL cell lines SNK-6 and YTS,up regulate the expression of Bax protein,down-regulate the expression of BCL-2 protein,and down-regulate the expression of CCL22 protein mediated by FOXO3.Animal experiment shown that the baicalin can inhibit tumor growth.Baicalin can inhibit the growth and induce apoptosis of ENKTCL cells through FOXO3/CCL22signaling pathway.